The Effect And Mechanism Of Inhibiting G6PD Expression In Human Gastric Cancer SGC-7901by RNA Interference

BACKGROUND:Gastric cancer is one of the most common tumors in China and around the world, with a significant mortality. Although much progress has been made in diagnoses and treatments, the prognosis of gastric cancer is still poor with an estimated5-year survival rate of less than25%. Glucose-6-phosphate dehydrogenase (G6PD) is the first rate-limiting metabolic enzyme of the pentose phosphate pathway that produces NADPH and R5P. NADPH is indispensible for maintaining the redox equilibrium inside cells and protecting them from oxidative stress triggered by oxidant agents. R5P is one of the most important materials of nucleic acid synthesis. Thus, sufficient NADPH and R5P are necessary for cell dividing and proliferating, especially for the tumor cells with a rapid proliferation rate. The roles of G6PD in tumor are becoming the hot spot of tumor researches. Now it is confirmed that G6PD has a high expression in many tumors and plays an important part in the tumor growth and progress. However, the expression condition and effect in gastric cancer are still unclear. The tumor suppressor gene p53plays a major role in the protection of cells from DNA damage. p53makes these cells arrested in G1, allowing cells time to repair the damage in advance of S phase. The failure of the repairing will trigger apoptosis to prevent those cells with genetic mutations and chromosome aberrations from reproduction, which could lead to tumorigenesis. Recent researches have found crosstalk between p53and redox status in cells. p53was considered to be able to strengthen the pentose phosphate pathway. These facts give us a clue that there should be interactions between p53and G6PD which may bring great influence to tumor generation and development.OBJECTIVE:To investigate the role of G6PD in gastric cancer progression and the possible mechanisms. METHODS:1. Immunohistochemistry (IHC) was performed to detect the expression level of G6PD and mutation-type p53(mt-p53) in24cases of human gastric cancer and paired normal tissues (6-cm distances from the cancer). Then use the same method to detect the expression of G6PD and mt-p53in167specimens of gastric cancer tissues, in order to analyze the relationship of G6PD expression with clinicopathological features, prognosis and mt-p53expression.2. Design and synthesize three chemical modified small interference RNAs (siRNA) with different sequences for G6PD. According to the inhibition effect of the G6PD mRNA, choose the most specific siRNA to construct the G6PD siRNA plasmid (pGPU6/GFP/Neo-G6PD shRNA).3. Transfect the pGPU6/GFP/Neo-G6PD shRNA into gastric cancer SGC-7901cells with lipofectamine2000. All cells were divided into three groups, which were black control group (untransfected cells), negative control group (cells transfected with the pGPU6/GFP/Neo-shNC) and transfection group (cells transfected with pGPU6/GFP/Neo-G6PD shRNA). Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and western blot were performed to detect the expression of G6PD and p53mRNA and protein. Fluorescence probe was used to detect the expression of ROS and colorimetry for GSH. Cell proliferation was recorded with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. And the cell cycle phase distribution was assessed by flow cytometry. Induce oxidative stress by H2O2to observe the change of the cell sensitivity to apoptosis in such stress.RESULTS:1. The expression of G6PD and mt-p53protein in gastric cancer tissues were significantly higher than that in normal gastric mucosa tissues (P<0.05). Expression condition of G6PD was significantly associated with the size of tumor, depth of invasion, numbers of lymph node metastasis and TNM stage. The expression of G6PD and mt-p53was significantly positive correlated in gastric cancer tissues (r=0.528, P<0.01). In univariate analysis, the survival time of gastric cancer patients with strong expression of G6PD is much shorter than that with weak expression of G6PD. In Cox multivariate analysis, expression of G6PD and mt-p53were both independent prognostic factors.2. The three siRNAs:G6PD-homo-318, G6PD-homo-913and G6PD-homo-1727, all successfully inhibit the expression of G6PD mRNA in SGC-7901cells. While the best effect belongs to G6PD-homo-318whose interference efficiency is as high as86.61%. So the pGPU6/GFP/Neo-G6PD shRNA that contains the specific sequences of G6PD-homo-318has been constructed and confirmed by restriction enzyme digestion and sequencing.3. Compared to negative control group, the mRNA and protein level of G6PD in transfection group were inhibited by72.57%and63.96%(P<0.05), respectively. Compared to blank control group and negative control group, transfection group has a decreased GSH level (P<0.05) and elevated ROS level (P<0.05). The growth of cells was significantly decreased in transfection group (P<0.05). Moreover, specific G6PD siRNA significantly increased the number of cells in the pro-G0/G1phase and G0/G1phase (P<0.05), decreased the number of cells of in the S phase and G2/M phase (P<0.05), and decreased the proliferation index (P<0.05). The expressions of p53mRNA and protein level in transfection group were increased (P<0.05). The sensitivity to apoptosis induced by oxidative stress increased (P<0.05). CONCLUSION:1. G6PD is highly expressed in gastric cancer and the expression of G6PD and mt-p53protein was significantly positive correlated. Overexpression of G6PD is closely related with progression of gastric cancer, and might be regarded as one of the factors of prognosis.2. PGPU6/GFP/Neo-G6PD shRNA can efficiently inhibit the expression of G6PD mRNA and protein in gastric cancer SGC-7901cells.3. Silencing of G6PD expression in gastric cancer SGC-7901cells led the redox state in SGC-7901cells to offset to the oxidation direction, inhibited the cells growth and proliferation, and improved the sensitivity to oxidative stress-induced apoptosis. Meanwhile, silencing the expression of G6PD can increased the expression of p53mRNA and protein in SGC-7901cells.